The present invention relates to a sialyltransferase and to a DNA encoding the same. More particularly, the present invention relates to an enzyme which synthesizes ganglioside GM3 by transferring sialic acid to a galactose residue of lactosylceramide and to a DNA encoding the enzyme.
Human myelogenous leukemia cell line HL-60, which is a cell line that has acquired the ability of infinite proliferation as a result of tumorigenic transformation, is used generally and widely as a model for leukemia cells (Collins, S. J. Gallo, R. C., and Gallagher, R. E., Nature (London), 270, 347-349 (1977); Collins, S. J., Blood, 70, 1223 (1987)). The cell line does not differentiate even after continued cultivation and continues to proliferate while it remains as undifferentiated cells. However, when cultivation is continued with addition of phorbol ester, which is widely used as a differentiation inducer, the cell line stops the proliferation of cells and takes an appearance similar to that of monocytes or macrophages. This indicates that differentiation has been induced. It has been reported that during this process, the amount of GM3, which is a kind of ganglioside, increases considerably (Nojiri, E., Takaku, F., Tetsuka, T., and Saito, M., Blood, 64, 534-541 (1984)), and when the ganglioside GM3 is added exogenously, the cell line shows the same change as that observed with the addition of phorbol ester, i.e., the cells undergo monocytic differentiation (Saito, M., Terui, Y., and Nojiri, H., Biochem. Biophys. Res. Commun., 132, 223-231 (1985)). Also, it has been proved that in this differentiation process, GM3 itself has an activity of inducing differentiation (Nojiri, H., Takaku, F., Miura, Y., and Saito, M., Proc. Natl. Acad. Sci. U.S.A., 83, 782-786 (1986)), and that chemically synthesized Gm also induces differentiation (Sugimoto, M. and Ogawa, T., Glycoconj. J., 2, 5-9 (1985); Saito, M., Nojiri, H., Ogino, H., Yuo, A., Ogura, H., Itoh, M., Tomita, K., Ogawa, T., Nagai, Y., and Kitagawa, S., FEBS Lett., 271, 85-88 (1990)).
On the other hand, it has been elucidated that sialic acid-containing glycolipids, in particular ganglioside, bear important functions in various biological phenomena and not only its functions but also its biosynthesis are being clarified. In vertebrates, many gangliosides (ganglio-series gangliosides) have a common precursor, GM3, which has the simplest structure among major gangliosides and the GM3 synthesis affords a basis for the biosynthesis of gangliosides which have major functions.
As described above, ganglioside GM3 itself participates in the proliferation/differentiation of cells and tissues and it is suggested that the ganglioside GM3 is a precursor for a group of higher gangliosides having various functions in vertebrates.
GM3 has been considered to be synthesized from lactosylceramide by transfer of sialic acid to the galactose residue in lactosylceramide by CMP-sialic acid:lactosylceramide sialyltransferase (CMP-NeuAc: Galβ1-4Glcβ1-1′Cerα2,3-sialyltransferase; SAT-1). However, neither the transferase from mouse and human has been isolated nor the genes thereof have been identified.
Enzymes which transfer sialic acid through an α2-3 ketoside bond are described in, for example, Wienstein et al., J. Biol. Chem., 257, 13835 (1982); Gillespie et al., Glycoconj., 7, 469 (1990); Gillespie, W., Kelm, S, and Paulson, J C., J. Biol. Chem., 267, p 21001-21010 (1992); Lee, Y C., Kojima, N., Wada, E. Kurosawa, N., Nakaoka, T., Hashimoto, T. and Tsuji, S., J. Biol. Chem., 269, p 10028-10033 (1994); Kim. Y J., Kim, K S., Kim, S H., Kim, C H., Ko, J H., Choe, I S., Tsuji, S, and Lee, Y C., Biochem. Biophys. Res. Commun., 228, p 324-327 (1996); and JP-A 5-336963. However, none of the enzymes is known to be involved in the synthesis of GM3 or shows an enzyme activity of transferring sialic acid to lactosylceramide through an α2-3 ketoside bond. Sandhoff, K. et al. presume that α2-3 sialyltransferase (SAT4) is identical with the enzyme which synthesizes GM3 (J. Biol. Chem., 268, 5341 (1993)). However, this is a presumption based on an indirect method, which fails to support that the enzymes are identical to each other as a substance.
In spite of various attempts which have been made in order to elucidate and control its biosynthesis according as the clarification of importance of ganglioside GM3 proceeds, the above-mentioned sialyltransferase, which relates closely to the synthesis of GM3, has not been isolated yet from mouse and human because of difficulty in preparing the enzyme protein and, hence, neither its gene expression control mechanism has been clarified yet nor its proteo-chemical or enzymological analysis has been performed successfully.